To test the hypothesis that beta-cell M3 muscarinic receptors might facilitate glucose-dependent insulin release and lead to improved glucose tolerance, we generated transgenic mice that selectively overexpress M3 receptors in pancreatic beta-cells. To ensure that M3 receptors were selectively expressed by pancreatic beta-cells, transgene expression was placed under the control of a 0.65 kb fragment of the rat insulin promoter II. We recently reported the generation and analysis of transgenic mice that expressed a modified version of the M3 receptor (M3-del-i3) in pancreatic beta-cells (the central portion of the third intracellular loop was deleted in this construct; Gautam et al. Cell Metab. 3, 449-461, 2006). Strikingly, these mutant mice showed a pronounced increase in glucose tolerance and insulin release, and were resistant to diet-induced glucose intolerance and hyperglycemia. Radioligand binding studies showed that the M3-del-i3 receptor was expressed at very high levels in beta-cells (at least 30-fold higher than the levels of endogenous beta-cell M3 receptors).[unreadable] We recently extended these studies by generating transgenic mice that selectively overexpress the full-length M3 receptor in pancreatic beta-cells. Moreover, we also obtained transgenic mice that selectively overexpress a mutant version of the M3 receptor containing the Gln490Leu point mutation in pancreatic beta-cells. This point mutation leads to M3 receptor signaling even in the absence of an activating ligand. We selected transgenic lines that overexpressed the full-length M3 receptor and the Gln490Leu mutant receptor at similar levels (about 10-fold higher than the levels of endogenous beta-cell M3 receptors). The phenotypic analysis of these mutant animals is currently in progress. In preliminary experiments, both transgenic lines tested showed greatly improved glucose tolerance and enhanced insulin release. Besides the physiological analysis of these mutant mice, we are currently also carrying out a series of biochemical studies to determine to which extent different intracellular signaling pathways are altered in these animals. The preliminary results that we obtained so far support the concept that enhanced signaling through beta-cell M3 receptors can improve glucose homeostasis and that strategies aimed at stimulating this pathway might become clinically relevant in the treatment of type 2 diabetes. [unreadable] We recently demonstrated that treatment of the MIN6 mouse insulinoma cell line with muscarinic agonists causes a pronounced increase in insulin release. We also showed that this response was accompanied by a striking increase in intracellular calcium levels. Interestingly, treatment of MIN6 cells with M3 receptor siRNA greatly reduced both the muscarinic agonist-induced increase in insulin release and the elevation of intracellular calcium levels. This observation strongly suggests that both responses are predominantly mediated by the M3 receptor subtype, similar to what we found with native beta cells. The MIN6 mouse insulinoma cell line therefore represents an excellent in vitro model system to dissect the molecular pathways leading to M3 receptor-mediated insulin release. By using siRNA technology, we started to explore the molecular nature of the GRK (G-protein-coupled Receptor Kinase) and RGS (Regulator of G Protein Signaling) proteins that modulate M3 receptor function in these cells. We found, for example, that MIN6 cells express several RGS proteins at relatively high levels. RGS proteins are known to act as inhibitors of signal transduction cascades initiated by G-protein-coupled receptors (GPCRs) because of their ability to increase the intrinsic GTPase activity of heterotrimeric G proteins. This GTPase accelerating activity leads to G protein deactivation and promotes desensitization. Recent studies suggest that signaling through the M3 receptor is also under inhibitory control by various RGS proteins. We therefore started to use of siRNA technology to knock down specific RGS proteins in MIN6 cells. Interestingly, preliminary studies indicate that certain RGS proteins expressed by MIN6 cells exert an inhibitory effect on muscarinic agonist-induced insulin release and increased intracellular calcium levels. These findings suggest that agents that can inhibit the function of certain RGS proteins may become therapeutically useful in enhancing beta-cell M3 receptor function in type II diabetes.